Infection of a cell by HIV-1 proceeds through a series of steps, each of which defines a potential target for antiviral drug intervention. Current drugs target key viral activities catalyzed by the protease, reverse transcriptase, and integrase enzymes, but emergence of drug resistance calls for continual development of new drugs with novel modes of action. HIV-1 depends on interactions with numerous cellular proteins as it replicates and maraviroc, a drug that disrupts the interaction between the viral envelope glycoprotein and cellular surface CCR5 receptor, is an approved inhibitor of a virus-cell interaction that blocks the ability of the virus to enter the cell. Also crucial to HIV-1 replicatin is the ability for the preintegration complex intermediate that houses the integrase and reverse transcript to access the chromosomes inside the cell nucleus, where viral DNA integration takes place. Lentiviruses like HIV-1 use an energy-dependent process to transport their preintegration complexes through nuclear pore complexes that riddle the biphasic nuclear envelope, though the molecular mechanisms underlying HIV-1 nuclear transport are poorly understood. Work supported by this renewal grant application identified numerous cellular proteins as potential critical cofactors of HIV-1 replication and a significant number of these, including transportin-3, nucleoporin 153, and nucleoporin 358 were accordingly implicated in preintegration complex nuclear import due to their known functions within the cell. Subsequent work indeed confirmed this contention, though it remains unclear how these and other cellular and viral proteins precisely dictate HIV-1 preintegration complex nuclear import. Using numerous virology, biochemical, and genetic approaches, the work proposed herein will determine the mechanisms of HIV-1 nuclear import, focusing on novel protein interactions between the virus and host cellular components that are essential for the process. Such discoveries will define new targets for the development of inhibitors that block critical HIV-host interactions, which would be expected to increase the breadth of future antiviral armaments in the ongoing battle to control the spread of HIV/AIDS. PUBLIC HEALTH RELEVANCE: Despite HAART successes, drug resistance emerges due to the inherent viral genetic barrier required to build resistance to any given compound plus associated toxicity of compound use, which significantly affects patient compliance. These observations highlight the ongoing need to develop new antiviral inhibitors, and crucial interactions between HIV-1 and cellular cofactors have been highlighted in recent years as novel drug targets. This proposal will uncover novel details of how the HIV-1 preintegration complex, a critical viral replication intermediate, accesses the cellular nucleus where viral integration occurs, which will define novel targets for antiviral drug development moving forward.